multiplex qPCR can be very difficult to fully optimize, although the companies will tell you it's possible. I ended up running my duplex assay separately as optimizing the multiplex was taking too long.

I would try optimizing the assay on its own first then try optimizing the multiplex.

When I used probes instead of just Sybr green qPCR I found that using a larger reaction volume of 20ul rather than 8-10ul helped.

Also, you will want to use NCBI's blastn function to search your IC template, primers, and probe against the genome/exome of your organism to look for non-specific binding. there will need to be multiple mismatches, especially on the 3' end of the primers. if the 3' end can sit bind then it can amplify. 23S might be too conserved. if this does not work then I would recommend using a fully exogenous template like luciferase RNA as your internal control.

Designing an internal control (IC) for multiplex qPCR requires careful consideration of specificity, compatibility, and functionality.

Potential Issues:

If the sample contains bacterial DNA, the 23S rRNA primers might amplify it, leading to false IC signals.

Solution: BLAST your primers/probe against common contaminants (e.g., E. coli, plant chloroplasts) to ensure no cross-reactivity.

If cross-reactivity is a risk, consider redesigning the primers/probe to target a fully synthetic sequence (e.g., from a non-natural plasmid or phage DNA).

Your IC primers (degenerate at Y = C/T) may bind unintended sequences.

Recommendation:

Use non-degenerate primers for better specificity.

Test the IC in no-template controls (NTC) and DNA-free samples to confirm no amplification.

If amplification occurs in negative samples, redesign the IC to avoid endogenous sequences.

IC Concentration Optimization:

The IC should be low enough to avoid competition with targets but high enough to detect inhibition.

Start with ~50–100 copies/reaction and adjust based on Cq values (ideally Cq ~25–30).

Multiplex Compatibility:

Ensure the Cy5 probe does not cross-talk with FAM/VIC/ROX (check instrument filters).

Confirm no primer-dimers form between IC and target primers.

Inhibition Testing:

Spike IC into diluted samples to mimic inhibition (e.g., with humic acid or EDTA) and verify Cq shifts.

Use a plant-specific gene (e.g., lectin or zein) as an endogenous control for extraction efficiency.

For exogenous ICs, consider:

Lambda phage DNA (non-food background).

Artificial sequences (e.g., from Arabidopsis pseudogenes).

Validation Steps:

Test IC in 100+ samples (including GMO-negative and -positive matrices).

Check for Cq consistency (SD < 0.5 cycles across runs).

Confirm no impact on target sensitivity/LOD.

Final Recommendation Your IC could work, but:

BLAST the primers/probe to rule out off-target binding.

Test in NTCs and DNA-free samples to confirm specificity.

Optimize concentration to avoid competition.

Consider a fully synthetic IC if cross-reactivity is suspected.